Bottom Line:
Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active.This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence.A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.

ABSTRACTPalC, distantly related to Saccharomyces cerevisiae peripheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulans pH signalling proteins, contains a Bro1 domain. Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active. PalC recruitment to these structures is dependent on the seven transmembrane domain (7-TMD) receptor and likely pH sensor PalH. PalC is a two-hybrid interactor of the ESCRT-III Vps20/Vps32 subcomplex and binds Vps32 directly. This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence. In contrast, Arg442Delta impairs Vps32 binding and prevents PalC-GFP recruitment to cortical structures. pH signalling involves a plasma membrane complex including the 7-TMD receptor PalH and the arrestin-like PalF and an endosomal membrane complex involving the PalB protease, the transcription factor PacC and the Vps32 binding, Bro1-domain-containing protein PalA. PalC, which localizes to cortical structures and can additionally bind a component of ESCRT-III, has the features required to bridge these two entities. A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.

fig03: PalC two-hybrid interactions with ESCRT-III.A) PalC interacts with Vps32. The indicated GAL4 DNA-binding domain (DBD) and activation domain (AD) combinations were tested in Saccharomyces cerevisiaeY187 using the β-galactosidase filter lift assay. B) The indicated DBD and AD protein fusions were tested for two-hybrid interactions using quantitative β-galactosidase assays (given as enzyme units; SE, standard error). The reported PalA–Vps32 interaction is used here as a positive control. C) Network of PalC interactions tested in this work. The scheme summarizes data obtained using β-galactosidase quantitative (Figure 3B) and filter lift (not shown) assays with S. cerevisiaeY187 and the -Leu, -Trp, -His, -Ade QSM system (Figure S1) with S. cerevisiaeAH109. Arrows and ‘T’ symbols denote positive and negative two-hybrid interactions for a given prey-to-bait combination, as indicated. Those combinations that are not indicated have not been tested (Figure S1). Readers should note that while we have tested every possible two-hybrid combination between PalC and ESCRT-III proteins, we have not analysed interactions of all class E Vps proteins included in the diagram with one another. However, each of these ESCRT-III proteins has been validated (i.e. shown to give a positive interaction) with at least one partner in one orientation, and PalC is competent for interactions both as prey and as bait.

Mentions:
We addressed the possibility of PalC/ESCRT-III linkage by systematically testing PalC interaction with all components of ESCRT-III and its accessory AAA ATPase Vps4 using two-hybrid analysis (Figures 3 and S1). This analysis required the characterization of A. nidulansorthologues of yeast Did2p [the fungal homologue of charged MVB protein 1 (CHMP1) (38,39)], Vps2p, Vps24p, Vps25p and Vps20p, which will be reported elsewhere. Aspergillus nidulansVps32 has been characterized previously (22). [Note that, as indicated in the legend for Figure 3, we have not systematically tested in two-hybrid assays all the possible interactions of these ESCRT proteins with one another, as has been reported for S. cerevisiae(40).]

fig03: PalC two-hybrid interactions with ESCRT-III.A) PalC interacts with Vps32. The indicated GAL4 DNA-binding domain (DBD) and activation domain (AD) combinations were tested in Saccharomyces cerevisiaeY187 using the β-galactosidase filter lift assay. B) The indicated DBD and AD protein fusions were tested for two-hybrid interactions using quantitative β-galactosidase assays (given as enzyme units; SE, standard error). The reported PalA–Vps32 interaction is used here as a positive control. C) Network of PalC interactions tested in this work. The scheme summarizes data obtained using β-galactosidase quantitative (Figure 3B) and filter lift (not shown) assays with S. cerevisiaeY187 and the -Leu, -Trp, -His, -Ade QSM system (Figure S1) with S. cerevisiaeAH109. Arrows and ‘T’ symbols denote positive and negative two-hybrid interactions for a given prey-to-bait combination, as indicated. Those combinations that are not indicated have not been tested (Figure S1). Readers should note that while we have tested every possible two-hybrid combination between PalC and ESCRT-III proteins, we have not analysed interactions of all class E Vps proteins included in the diagram with one another. However, each of these ESCRT-III proteins has been validated (i.e. shown to give a positive interaction) with at least one partner in one orientation, and PalC is competent for interactions both as prey and as bait.

Mentions:
We addressed the possibility of PalC/ESCRT-III linkage by systematically testing PalC interaction with all components of ESCRT-III and its accessory AAA ATPase Vps4 using two-hybrid analysis (Figures 3 and S1). This analysis required the characterization of A. nidulansorthologues of yeast Did2p [the fungal homologue of charged MVB protein 1 (CHMP1) (38,39)], Vps2p, Vps24p, Vps25p and Vps20p, which will be reported elsewhere. Aspergillus nidulansVps32 has been characterized previously (22). [Note that, as indicated in the legend for Figure 3, we have not systematically tested in two-hybrid assays all the possible interactions of these ESCRT proteins with one another, as has been reported for S. cerevisiae(40).]

Bottom Line:
Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active.This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence.A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.

ABSTRACTPalC, distantly related to Saccharomyces cerevisiae peripheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulans pH signalling proteins, contains a Bro1 domain. Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active. PalC recruitment to these structures is dependent on the seven transmembrane domain (7-TMD) receptor and likely pH sensor PalH. PalC is a two-hybrid interactor of the ESCRT-III Vps20/Vps32 subcomplex and binds Vps32 directly. This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence. In contrast, Arg442Delta impairs Vps32 binding and prevents PalC-GFP recruitment to cortical structures. pH signalling involves a plasma membrane complex including the 7-TMD receptor PalH and the arrestin-like PalF and an endosomal membrane complex involving the PalB protease, the transcription factor PacC and the Vps32 binding, Bro1-domain-containing protein PalA. PalC, which localizes to cortical structures and can additionally bind a component of ESCRT-III, has the features required to bridge these two entities. A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.